Cilia driven flow is a key element in widely accepted models for left-right patterning. Yet we still do not know how cilia or cilia-driven flow give riseto the asymmetric expression of nodal. The long-term goal is to determine how the LR axis is established and how the asymmetric information generated is utilized to direct organ morphogenesis. The next step towards realizing this goal is to identify how signaling downstream of cilia establishes asymmetric nodal expression. The central hypothesis underlying this proposal is that cilia-driven fluid flow in Kupffer's vesicle (KV) signals through he PC1L1/PC2 channel complex to repress transcription of charon on the left side of the embryo. This hypothesis is based in part on preliminary data demonstrating that the zebrafish nodal gene southpaw (spaw) is not expressed in cells lining KV and is thus not the direct target of signaling downstream of flow. Instead, loss of PC2 activity affects the asymmetric expression of the Nodal inhibitor charon at KV. PC2 is thought to be the flow sensor in LR patterning, suggesting that charon is the target of flow-generated signaling. Thus, charon expression at higher levels on the right side of the embryo would inhibit Spaw signaling on the right, allowing Spaw to signal preferentially to the left side of the embryo. The rationale for this project is tha upon completion, this work will have provided the missing links between cilia and the control of asymmetric nodal expression. This information is crucial to fully understand how signaling downstream of cilia may participate LR patterning, disruptions in which cause congenital heart disease (CHD). The central hypothesis will be tested through three specific aims: 1) Determine how signals downstream of the non-specific cation channel PC2 regulate charon asymmetry. 2) Determine if PC1L1 signals are required to regulate charon asymmetry. 3) Identify the transcriptional mechanisms that generate asymmetric charon expression. In Aim 1, the localization and activity of PC2 required for generating charon asymmetry will be explored. Calcium imaging with KV specific promoters will be used to determine if PC2 is creating calcium signals in response to flow at the right place and time to affect charon expression. In Aim 2, the role for PC1L1 in zebrafish will be confirmed. Experiments to test whether the C-terminal tail is cleaved in response to flow in order to regulate transcription will be tested. In Aim 3, the smallest enhancer fragment capable of driving asymmetric expression of charon will be determined and used to identify transcription factors and signaling pathways involved in asymmetric charon expression. The proposed research is significant because it is the first step towards understanding how flow influences signaling that participates in establishment of left-right patterning. The approach is innovative, as it is a directed approach utilizing the tools and genetics available in the zebrafish system, to tackle a difficult but critical question in left-rigt patterning that is not currently being explored fully. Ultimately this work will identify signaling networks participating in the establishment of the LR axis and will provide new targets to investigate as factors underlying CHD. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public heath because our identification of the signaling pathways involved in establishing left-right patterning will provide important new targets to investigate as factors underlying congenital heart disease (CHD). As surgical intervention improves, there is a growing need to identify the important causative factors for CHD, so that adults born with CHD can receive appropriate health care and proper testing to determine their risk of having a child with CHD. Thus, the proposed research is relevant to the NIH's mission to develop the fundamental knowledge needed to extend healthy life and reduce the burdens of illness.